Streptococcus pyogenes or group A Streptococcus (GAS) is the agent of streptococcal pharyngitis, infections of the skin and soft tissue, and life-threatening invasive infections. GAS infections and post-infectious sequelae, primarily in the form of rheumatic heart disease, account for 500,000 deaths annually in addition to morbidity and economic losses. A vaccine is not yet available and antibiotic treatment fails or is followed by relapse in 10 to 20 percent of pharyngitis cases. The refractory nature of GAS infection may reflect the co-evolution of GAS with human beings and its exquisite adaptation for survival in the human pharynx, its primary environmental niche. We and others have observed entry of GAS into human epithelial cells in vitro. While intracellular bacteria are eventually killed, we have found that GAS co-toxins streptolysin O and NADase inhibit GAS internalization and prolong intracellular survival, effects that may contribute to antibiotic treatment failure, relapse, and prolonged carriage. The goal of this proposal is to determine how SLO and NADase block internalization of GAS by pharyngeal epithelial cells and prevent effective intracellular killing. Specific aims are (1) to characterize internalization of GAS by pharyngeal epithelial cells, (2) to determine the specific roles of SLO and NADase in blocking internalization, and (3) to define the molecular mechanisms by which GAS avoids or escapes intracellular killing. In preliminary studies, we have found striking effects of SLO and NADase on the fate of GAS during interaction with keratinocytes. We have assembled the necessary molecular tools, cell culture models, and imaging techniques to decipher how these toxins and host cellular mechanisms determine the outcome of this critical pathogen-host interaction at the pharyngeal epithelium. The results will inform new approaches to prevention and treatment of GAS infection. Project Narrative: Group A Streptococcus or GAS is responsible for millions of cases of streptococcal sore throat and approximately 10,000 cases of life-threatening invasive infections annually in the United States, and rheumatic fever, a complication of GAS infection, is a leading cause of often fatal heart disease in developing countries. The objective of this project is determine how cells lining the human throat ingest and kill GAS, thereby blocking GAS infection, and how two GAS toxins act to counteract this host defense mechanism. Understanding these interactions may lead to novel strategies for prevention or treatment of GAS infections.